title: Identification of PP2A as a novel interactor and regulator of TRIP-Br1.
abstract: TRIP-Br proteins are a novel family of transcriptional coregulators involved in E2F-mediated cell cycle progression. Three of the four mammalian members of TRIP-Br family, including TRIP-Br1, are known oncogenes. We now report the identification of the Balpha regulatory subunit of serine/threonine protein phosphatase 2A (PP2A) as a novel TRIP-Br1 interactor, based on an affinity binding assay coupled with mass spectrometry. A GST-TRIP-Br1 fusion protein associates with catalytically active PP2A-ABalphaC holoenzyme in vitro. Coimmunoprecipitation confirms this association in vivo. Immunofluorescence staining with a monoclonal antibody against TRIP-Br1 reveals that endogenous TRIP-Br1 and PP2A-Balpha colocalize mainly in the cytoplasm. Consistently, immunoprecipitation followed by immunodetection with anti-phosphoserine antibody suggest that TRIP-Br1 exists in a serine-phosphorylated form. Inhibition of PP2A activity by okadaic acid or transcriptional silencing of the PP2A catalytic subunit by small interfering RNA results in downregulation of total TRIP-Br1 protein levels but upregulation of serine-phosphorylated TRIP-Br1. Overexpression of PP2A catalytic subunit increases TRIP-Br1 protein levels and TRIP-Br1 co-activated E2F1/DP1 transcription. Our data support a model in which association between PP2A-ABalphaC holoenzyme and TRIP-Br1 in vivo in mammalian cells represents a novel mechanism for regulating the level of TRIP-Br1 protooncoprotein.
authors.affiliation: Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Zhi Jiang
authors.initials: ZJ
authors.lastname: Zang
authors.forename: Lakshman
authors.initials: L
authors.lastname: Gunaratnam
authors.forename: Jit Kong
authors.initials: JK
authors.lastname: Cheong
authors.forename: Li Yun
authors.initials: LY
authors.lastname: Lai
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Hsiao
authors.forename: Eileen
authors.initials: E
authors.lastname: O'Leary
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Sun
authors.forename: Manuel
authors.initials: M
authors.lastname: Salto-Tellez
authors.forename: Joseph V
authors.initials: JV
authors.lastname: Bonventre
authors.forename: Stephen I-Hong
authors.initials: SI
authors.lastname: Hsu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D008163:Luminescent Measurements; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D000092468:Bromodomain Containing Proteins
chemical_list: D009687:Nuclear Proteins; D019319:Okadaic Acid; D054648:Protein Phosphatase 2; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; C485730:Sertad1 protein, mouse; D000092468:Bromodomain Containing Proteins